RU2159810C1 - Influenza virus strain a/47/peking/95/35 (h1n1) for production of live influenzal intranasal vaccine for babies - Google Patents

Abstract

FIELD: applied microbiology. SUBSTANCE: vaccinal strain A/47/Peking/95/35 (H1N1), which is reassortant, is obtained by cross-breeding of epidemic virus A/Peking/262/95 (H1N1) with cold-adapted temperature-sensitive virus A/Leningrad/134/47/57 (H2N2), which is attenuation donor. The strain is harmless for babies. It actively multiplies in developing chicken embryos at optimal temperature 33-34 C and is characterized by temperature sensitivity and cold adaptability. Reassortant inherits two genes from epidemic virus, which encode surface proteins (gemmagglutinin and neuraminidase), and six genes from attenuation donor encoding non-glycosylated proteins. In addition, the strain does non induce immune response in babies during intranasal injection. EFFECT: reliably enabled isolation of reassorted vaccinal strains from newly appearing epidemic viruses. 1 tbl

Description

 The invention relates to medical virology and can be used in healthcare for the prevention of epidemic influenza among children with a live intranasal influenza vaccine from strain A / 47 / Beijing / 95/35 (H1N1). The well-known strain A / 47 / Texas / 91/2/6 (H1N1) - prototype [1] - lost its antigenic relevance and as a result will not be able to cause a protective reaction during an influenza epidemic caused by a strain of influenza virus similar to A / Beijing / 262 virus / 95 (H1N1).

 The problem to which the invention is directed, is to obtain a vaccine strain of the actual antigenic variety based on the epidemic virus A / Beijing / 262/95 (H1N1).

 Vaccine strain A / 47 / Beijing / 95/35 (H1N1) was obtained by genetic reassortment of the epidemic virus A / Beijing / 262/95 (H1N1) with a cold-adapted strain A / Leningrad / 134/47/57 (H2N2) - attenuation donor - s subsequent selection in the presence of antisera to the virus A / Leningrad / 134/47/57 (H2N2). The attenuation donor is a cold-adapted temperature-sensitive strain of influenza virus A / Leningrad / 134/47/57 (H2N2), approved for the production of harmless live intranasal vaccines for children [2].

Using restriction analysis of DNA copies of the genes of the parent viruses and the reassorted vaccine strain, it was found that the vaccine strain A / 47 / Beijing / 95/35 (H1N1) inherited 6 genes encoding internal proteins (PB1, PB2, PA, NP, M, NS) from donor attenuation A / Leningrad / 134/47/57 (H2N2), and 2 genes encoding surface proteins - HA (hemagglutinin) and NA (neuraminidase) - from the epidemic virus A / Beijing / 262/95 (H1N1). Hemagglutinin in rtga is identical to virus A / Beijing / 262/95 (H1N1), to which it is completely neutralized by antiserum. The strain is temperature-sensitive (the difference in the rate of infectious activity at 33 ^o C and 40 ^o C is 7.0 lg EID ₅₀ / 0.2 ml) and cold-adapted (the difference in the rate of infectious activity at 33 ^o C and 25 ^o C is 3.0 lg EID ₅₀ / 0.2 ml).

 Thus, the presented vaccine strain A / 47 / Beijing / 95/35 (H1N1) is characterized by a combination of useful features necessary for the vaccine strain: antigenic specificity of the surface proteins of the epidemic virus A / Beijing / 262/95 (H1N1), the genome structure optimal for reassortant vaccine strains, temperature sensitivity and cold adaptation, which correlates with attenuation for humans, characteristic of an attenuation donor.

 The morphology of the strain is polymorphic, typical of the influenza virus.

STRAIN CHARACTERISTIC
Infectious activity during reproduction in developing chicken embryos at 33-34 ^o C for 48 hours - 8.5-9.0 lg EID ₅₀ / 0.2 ml

 Hemagglutinating activity -1: 512.

 The strain exhibits genetic stability of biological traits after 5 passages on chicken embryos (when using large infectious doses).

 A sample passport for the vaccine strain A / 47 / Beijing / 95/35 (H1N1) is attached.

 STRAIN A / 47 / BEIJING / 95/35 (H1N1) AREACTOGEN WITH INTRANASAL ADMINISTRATION FOR CHILDREN 3-14 YEARS.

Example. A group of 101 children aged 3-14 was vaccinated intranasally in a volume of 0.5 ml with the recombinant vaccine strain A / 47 / Beijing / 95/35 (H1N1) with an infectious activity of 8.5 1g EID ₅₀ / 0.2 ml. A group of children of the same age of 101 people received a placebo.

Strong temperature reactions with increasing temperature over 38.5 ^o C and moderate reactions with a short-term temperature increase in the range of 37.6-38.5 ^o C were not observed. Weak temperature reactions (up to 37.5 ^o C) in vaccinated were observed in 11 people (10.9% of cases). Weak temperature reactions (up to 37.5 ^o C) in the placebo group were observed in 15 people (14.9% of cases). The reactogenicity index (the difference in the percentage of average reactions in vaccinated vaccines and received placebo) was 0% (table 1).

 Thus, the vaccine strain A / 47 / Beijing / 95/35 (H1N1) in terms of reactogenicity meets the requirements for vaccine strains of Pharmacopoeia article FS 42-3353-97 for live influenza vaccine for intranasal use in children 3-14 years old.

PASSPORT STRAIN
1. The name of the strain is A / 47 / Beijing / 95/35 (H1N1).

 2. Series - Series 1.

3. The production method is recombination; characteristic of parent viruses:
a) epidemic virus - A / Beijing / 262/95 (H1N1);
b) donor of attenuation - A / Leningrad / 134/47/57 (H2N2).

 4. The number of passages - 6 in the process of recombination.

5. Characterization of the strain before lyophilization:
a) the optimal conditions for reproduction - 33 ^o C, 48 hours;
b) hemagglutinating activity - 1: 512;
c) infectious activity - 8.5 lg EID ₅₀ / 0.2 ml;
d) sensitivity to inhibitors - inhibitor-resistant;
d) the difference in the indicators of infectious activity at 33 ^o C and 40 ^o C - 7.0 lg EID ₅₀ / 0.2 ml;
e) the difference in the indicators of infectious activity at 33 ^o C and 25 ^o C - 3.0 lg EID ₅₀ / 0.2 ml;
g) the structure of the recombinant genome:
- genes from an epidemic virus - HA, NA;
- genes from an attenuation donor - RA, PB1, PB2, NP, M, NS.

6. Characterization of the strain after lyophilization:
a) date of lyophilization - September 14, 1998;
b) the volume of material in the ampoule is 1 ml;
c) the number of doses in a series is 3;
d) infectious activity - 7.5 lg EID ₅₀ /0.2 ml;
d) hemagglutinating activity - 1: 256.

 7. The recommended dilution for vaccination is 1: 3.

8. Antigenic specificity
a) hemagglutinin in rtga - identical to virus A / Beijing / 262/95 (H1N1), the antiserum of which is completely neutralized;
b) neuraminidase - identical to virus A / Beijing / 262/95 (H1N1) according to PCR restriction analysis of DNA copies of genes.

 9. Harmless to mice with subcutaneous and intraperitoneal administration - harmless.

 10. Bacteriological control of lyophilized material: date: September 14, 1998, the result is sterile.

 11. Control for the absence of extraneous viruses - extraneous viruses are absent.

LITERATURE
1. Strain A / 47 / Texac / 91/2/6 (H1N1) for the production of live influenza intranasal vaccine for children. RF patent 2077581 from 04.20.97. Published in the bulletin of inventions N 11 from 04/20/97.

 2. Alexandrova G.I., Klimov A.I. Live flu vaccine. - SPb .: Nauka. - 1994.- 151 p.

Claims (1)

 The strain of the influenza virus A / 47 / Beijing / 95/35 (H1N1) GISK N 394/5 (State Institute of Standardization and Control named after L.A. Tarasevich) used to obtain a live influenza intranasal vaccine for children.

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